Transmission of Alternaria macrospora in Cotton Seeds

Alternaria macrospora was isolated from seeds only after the natural opening of the bolls and exposure of seeds to an environment in which the fungus was present. The fungus lacks the ability to penetrate the boll wall and reach the seed site. Attempts to isolate the pathogen from seeds of immature bolls at different developmental stages failed. Internal infection by slow injection resulted in seed infection and partial shedding of the injected plant parts which was high in buds and decreased with the ripening to mature bolls. Severity of seed infection was not dependent on either inoculum level, boll physiological age or even if the boll itself was not diseased. Infection of flowers under field conditions caused flower shedding. Naturally infected seeds or inoculated seeds with inoculum levels of 100 spores/ml and above resulted in diseased cotyledons, the incidence of which was, for inoculated seeds, positively correlated with inoculum level. A small difference was observed between cultivars in susceptibility to artificial inoculation at the cotyledon stage. A. macrospora survived on commercial cotton seeds and on post-season plants left growing at the field edges. Survival in plant debris under field conditions was minimal and may only have a minor effect on field reinfestation.     

Phenols in Cotton Seedlings Resistant and Susceptible to Alternaria macrospora

In healthy cotton seedlings, stems have a lower phenol content than leaves, but resistant plants have an altogether relatively higher phenol content than susceptible plants. Phenols extracted from infected plants can inhibit the growth of A. macrospora in vitro. In cotton plants infected with A. macrospora, phenols are oxidized by polyphenoloxidase rather than peroxidase and catalase. The main oxidative activity was around the developing necrotic area but activity was detected far from, necrosis as well. Though pre–inoculation mechanical injuries operated the phenol oxidation mechanism in the plant, they neither prevented nor encouraged the increase in disease severity. Isozyme pattern showed that contribution of all participants in the pathological interaction to the oxidative mechanism occurred in the diseased plant. A negative linear correlation was found between polyphenoloxidase activity, phenol accumulation and resistance. This study suggests that the phenol oxidative mechanism, participates in cotton plant resistance to A. macrospora.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

Enzyme-Histochemical Identification of Lymphatic Vessels by Light and Backscattered Image Scanning Electron Microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.     

Morphology and Ultrastructure of Staphylococcal L Colonies: Light, Scanning, and Transmission Electron Microscopy

Scanning electron microscopy utilizing critical point drying was used in parallel with light and transmission electron microscopy to study L colonies produced by a stable L-phase variant of Staphylococcus aureus (AH24H).     

The CryoCapsule: Simplifying Correlative Light to Electron Microscopy

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high‐resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over‐expressing YFP‐langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.      

Scanning Electron Microscopy of Plant Roots

A glycol methacrylate infiltration and polymerization techniquewas used to prepare clover roots inoculated with Rhizobium forscanning reflection electron microscopy. Root hairs and epidermalcells were coated with many bacteria; some bacteria seemed tobe embedded in the wall surface. Root hair tips were often smoothbut some older root hair surfaces showed a fibrillar meshworkpattern. Small granules c. 0.18 µm diameter were presenton the root hair and epidermal cell walls. The root cap, someroot hairs, and some epidermal cells were covered by an amorphousfilm thought to be the mucigel.     

Scanning Electron Microscopy of Thermoplasma acidophilum

The scanning electron microscope was utilized to observe the morphology of the thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum. Upon examination of the surface morphology, the size and shape of this unusual mycoplasma revealed its similarity to the other mycoplasmas that have been investigated.     

Scanning Electron Microscopy of Bacterial Colonies

A technique is described for observing bacterial colony growth. Bacillus cereus, B. subtilis, and B. cereus var. mycoides were grown on strips of dialysis membrane layered on nutrient agar. Microcolonies of the organisms on strips were fixed in Formalin vapor in situ; the strips then were removed from the agar and secured to scanning microscope specimen stubs without markedly disturbing the cellular arrangement. Scanning electron micrographs clearly depict morphology of individual cells, as well as the spatial orientation of cells within the colony. This technique is reproducible, adaptable, and simple.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Correlative Photoactivated Localization and Scanning Electron Microscopy

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.     

Excavation of Boreholes by the Gastropod, Urosalpinx: An Analysis by Light and Scanning Electron Microscopy

Penetration of shell by the muricid gastropod, Urosalpinx cinereafollyensis, is accomplished by successive alternating periodsof (a) chemical activity by the accessory boring organ (ABO), and (b) rasping by the radula. This paper reports on the functionsof the radula and of the ABO in producing the characteristicgeometry of the borehole, andon the effects of radular teethand of the ABO secretion on the microscopic anatomy of the surfaceof the borehole during the process of shell-boring. Radulae of U. c. follyensis and the surfaces of incomplete boreholesin the shell of Crassoslrea virginica, Mytilus edulis, and Myawere examined by means of light and scanning electron microscopy.Hardness tests of radular teeth andshell of prey demonstratedthat marginal teeth are harder than rachidian teeth, and thatthe range of hardness of rachidianteeth overlaps that of thethree species of shell. Rasping is carried out by two, occasionallythree, of the five rachidiancusps. Rasping patterns are shallowand asymmetric. Rachidian teeth are worn to the base with use;marginal teeth wear onlyslightly as they are employed mainlyin feeding. The distance between the tips of rachidian cuspscorresponds with the interval between the parallel cusp tracesrasped by them in shell. During each rasping period, snailsscrape off about 1/10 to 1/5 of the surface of the chemicallytreated area of the bottom of the borehole. Dissolution of shell is accomplished by secretion from the secretorydisk of the ABO. With each application of the ABO,most or allof the radular marks of the previous rasping period are erasedby solution of a thin layer of shell. The pattern of etchingis specific for each of the species of shell studied. In oysterand mussel shell, initial solubilization occurs through theorganic, non-mineralized, prism sheaths, exposing prismaticforms shown by other workers to be distinctive for these species,and then proceeds into the organic-calcareous structure of individualprisms. Etching of Mya shell revealed no fundamental prismaticform. Shell-penetration includes dissolution of both organiccomplexes and CaCO₃ crystals. Shell-boring by this snail is principally a chemical process,and the geometry of the borehole is generally a reflection ofthe morphology of the ABO.     

Scanning Electron Microscopy of Ascospores of Schwanniomyces

Ascospores of the four recognized species of Schwanniomyces were examined by scanning electron microscopy. Spores of S. alluvius, S. castellii, and S. occidentalis, which were essentially identical, had abundant, long protuberances and wide, thin equatorial rings. The two known strains of S. persoonii differed from the other species as well as from each other. One strain had spores with a wide ring but only a few short protuberances; spores from the second strain were covered with craterlike depressions and had a narrow ring. Also examined were spores of Schwanniomyces hominis (=Saccharomyces rosei) which lacked a ring and were covered with short irregularly shaped protuberances, a finding consistent with the morphology of spores from other strains of S. rosei.     

Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Scanning Electron and Phase-Contrast Microscopy of Bacterial Spores

The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus, and Sarcina ureae have varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis, and B. licheniformis have relatively smooth surfaces.     

Hexamethyldisilazane for Scanning Electron Microscopy of Gastrotricha

We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 μm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermeila) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD.     

Cryo-Preservation of Roots for Scanning Electron Microscopy

Fully hydrated roots can be examined in the scanning electronmicroscope after cryo-preservation. Shrinkage associated withdehydration by freeze-drying or critical point drying, to whichroot hairs and secreted mucigel are particularly vulnerable,is avoided. Roots, Lepidium sativum, scanning electron microscopy, cryo-preservation, fully hydrated     

Scanning Electron Microscopy of Intact Trichoderma Colonies

Scanning electron microscopy revealed a clear distinction between hyphal types and enabled early detection of hyphal initiation. Stages in the photoinduced differentiation in Trichoderma leading to conidiation could thus be studied.